Accepted Manuscript

Effects of enzymatic treatment using response surface methodology on the

quality of bread flour

Mahsa Shafisoltani, Mania Salehifar, Maryam Hashemi

PII: S0308-8146(13)01460-X
DOI: http://dx.doi.org/10.1016/j.foodchem.2013.10.026
Reference: FOCH 14812

To appear in: Food Chemistry

Received Date: 9 April 2013

Revised Date: 4 October 2013
Accepted Date: 7 October 2013

Please cite this article as: Shafisoltani, M., Salehifar, M., Hashemi, M., Effects of enzymatic treatment using response
surface methodology on the quality of bread flour, Food Chemistry (2013), doi: http://dx.doi.org/10.1016/
j.foodchem.2013.10.026

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Effects of enzymatic treatment using response surface methodology on the

quality of bread flour

Mahsa Shafisoltania,* , Mania Salehifara , Maryam Hashemib

a
Department of Food Science and Technology , Azad University, Shahre ghods Branch , Tehran, Iran.
b
Biotechnology and Biosafety Department , Agricultural Biotechnology Research Institute , Karaj,

Iran.
8
Corresponding author : Mahsa Shafisoltani

Tell: 00982636602300 Fax:009886604300

Soltani89@ymail.com

Abstract:

Flour with low α-amylase activity needs to be supplemented with additional α –

amylase, but α –amylase added to weak flour can lead to decreased quality of the

dough. The objective of this study, was to evaluate the effects of glucose oxidase

(1-5 g/100g flour) and xylanase (1-3 g/ 100g flour) on the quality of bread flour

after optimization by additions of α–amylase. The effects of enzyme additions on

dough properties and bread quality parameters such as specific volume, shape,

texture and sensory attributes were determined by Response Surface Methodology

(RSM) using a central composite design. Results of RSM modeling showed that

glucose oxidase and xylanase improved the quality of bread and dough but effects

were dose dependent. In this work, the optimal dose of glucose oxidase and xylanase

were (30and20) ppm, respectively.

Keywords:

Wheat flour, α-amylase, Glucose oxidase, Xylanase, Response Surface Methodology

1. Introduction

Bread is the product of baking a mixture of flour, water, salt, yeast and other

ingredients (Whitehurst & Oort, 2010). An optimum bread making processes is one

1
that produces dough that rises well. This can be achieved by controlling amounts of

gas produced by the yeast, so that its release during the bread making process is

sustained to maintain elasticity of the dough to facilitate expansion during proving

(Whitehurst et al., 2010).

One of the major factors affecting gas production in dough is α- amylase enzyme

activity. (Wong & Robertson.,2002). Addition of α- amylase degrades the damaged

starch in wheat flour in bread dough into small dextrin, which allows the yeast to

work continuously during fermentation and produces CO2 gas. This serves to

improve volume and crumb texture in the final bread product. In addition, small

oligosaccharides and sugars such as glucose and maltose produced by these enzymes

enhance Maillard reactions that are responsible for browning of the crust and

development of an attractive flavor of the baked bread (Whitehurst et al., 2010).

Amylase activity of the flour is expressed by the falling number .A good flour has a

falling number between 200 and 250 seconds. Flours with a high falling number

due to minimal α- amylase activity cause problems in bread quality parameters

.Low α- amylase activity in flour leads to low dextrin production and poor gas

production. This in turn results in inferior quality bread with reduced size and

poor crust color. So, there is a need for flours with low amylase activity to be

supplemented with α- amylase. These can be added in the form of fungal

amylase (Whitehurst et al.,2010).

However adding fungal α- amylase to flour that has a weak gluten network leads

to a sharp decrease in stability of the dough ,and this makes it weaker and

results in dough with a sticky texture. Sticky dough causes handling problems and

affects its capacity to rise, which may lead to rejection of a batch. These changes do

not allow dough to retain the gases fermentation (Pritchard and Peter, 2010). For

2
solving dough problems, the combination of xylanase and glucose oxidase enzymes

can be added to flour. Xylanase and glucose oxidase contributing to better

structure of gluten and its formation (Almeida & Chang , 2012).

Glucose oxidase canbe used for strengthening of the dough. Glucose oxidase has a

gluten strengthening effect by inducing the formation of protein- protein bonds

that strengthen the protein network and thereby strengthen and stabilize the

dough (Whitehurst et al. , 2010).Glucose oxidase catalyzes the oxidation of

glucose to the gluconolactone with the concomitant reduction of the oxygen to

hydrogen peroxidase (Hansen & Stougaard, 1997). Five mechanisms have been

reported in the literature concerning the effects of the sugar oxidase enzymes,

as follows: (i) generation of disulfide bridges between the proteins (Joye, lagrrain ,

& Delcour, 2009); (ii) formation of di tyrosine (Hanft and Koehler, 2005); (iii)

blocking the effect of glutathione (Forman,2004); (iv) gelling of the soluble

arabinoxylans in the flour (Forman, 2004; Joye et al.,2009); and (v) altering the

equilibrium between the different enzyme systems present in the dough (Garcia,

Rakotozafy, & Nicolas, 2004).

Xylanase is used in the backing industry to stabilize dough, to make it flexible

andto improve gluten strength (Collins,Hoyoux,Dutron,Georis,Genot,Daurvin,2006).

Xylan is the principal hemicellulose which is the major plant cell wall

polysaccharide component. Arabinoxylans are important in the formation of

gluten and other networks in dough, and it has been reported that the non-water

extractable arabinoxylans have a negative effect on bakery products, whereas

the water extractable forms of medium to high molecular weight, have a

beneficial effect (Courtin,Gelder,&Delcour,2001).

3
Xylanases randomly hydrolyze the β-1,4-glycosidic bonds of xylan to produce

xylooligomers of different lengths (Viikari , Alapuranen , Puranen, Vehmaanpera

& Siika- Aho, 2007 ). Xylanases have added to dough to accelerate the process of

producing bread by helping to breakdown polysaccharides in the dough

(Courtin & Delcor, 2002). Enzyme activity is effective in dough by cleaving

arabinoxylan chains and, thus modifying their function (Primo- Martin, Wang,

Lichtendonk, Plijter&Hamer,2005).

Steffolani et al., studied the effects of the combination of glucose oxidase, a-

amylase and xylanase on dough properties and bread quality apart from the initial

grain origin a-amylase activity (Steffolani,Ribotta,Perez,Leon., 2012). They

concluded that combination of intermediate levels of the above mentioned enzymes

results in dough with low stickiness and a bread with 40% higher specific volume in

comparison to the sample without enzyme.

The level of α-amylase is a key quality parameter in bread making. Before adding α-

amylase to flour , initial grain origin amylase activity should be measured by falling

number at first, because adding more or less than optimum level of this enzyme to

flour results in inferior quality bread .In this study, α-amylase activity of the flour

was low. To compensate for the deficiencies of the grain, α- amylase was added to

flour. Adding fungal α- amylase to flour leads to a sharp decrease in stability

of the dough , For solving dough problems, the combination of xylanase and glucose

oxidase enzymes was added. So, the objective of this study was to evaluate the

effect of adding the combination of glucose oxidase and xylanase enzymes on

the quality of dough properties and bread quality parameters using central

composite rotational design, after optimization of α- amylase activity of flour.

2. Materials and methods

4
2-1- Materials

All the materials used were provided by the manufactures. Commercial wheat

flour (Tak karaj co - Tehran- Iran) was used. It presented moisture, proteins (N×5.7),

ash and gluten content of 13.2 g, 11, .580 g 27 g /100 g flour, respectively. And the

Falling Number was 603 s. Fungal alpha amylase (Millbo Co-Italy) derived from

Aspergillus oryzae the activity of enzyme preparation was 120000 skb/min.

Glucose oxidase (Millbo co-Italy) derived from Aspergillus Oryzae the activity of

enzyme preparation was 10000 u/g . Xylanase (Millbo co-Italy) derived from

Aspergillus oryzae the activity of enzyme preparation was 20000 u/g.

2- 2 -Methods

2-2-1- Characterization of wheat flours

The determination of moisture , protein, ash and gluten were realized using

AACC methods 44-10.01, 46-13.01, 08-01.01 and 38-12-02 (2010), respectively,

falling number (FN) for measuring grain origin amylase activity and fungal falling

number (FFN) for measuring fungal amylase activity were analyzed according to

AACC method 56-81-03(2012) on the Infracont 5000 equipment. FN test is done at

boiling temperature but FFN test is measured at the temperature of 90° c. To the α-

amylase comes from grain origin, FN analysis is done on raw material at the

beginning of tests while following the addition of fungal α- amylase the result of the

final product should be controlled by FFN.

Evaluation values of the natural amylase activity and fungal amylase activity

according to operating instruction were as follows: Fungal falling number (FFN)

value of 170 s or longer indicate a low fungal amylase enzyme activity. FFN Values

below 110 s indicate high level of fungal amylase enzyme activity and values

between110 to 170 indicate normal level of fungal amylase enzyme activity.

5
Falling number (FN) value of 280 or longer indicate a low natural amylase enzyme

activity. Values below 250 s indicate high level of natural amylase enzyme activity.

2-2-2- Determination of mixing behavior of the flour

A Brabender farinograph (Duisburg, Germany) with a 50 g stainless steel bowl

was used to evaluate the impact of the enzyme addition according to the

approved method 115/1 (ICC,1992) the following parameters were determined in

the farinograph analysis : water absorption, dough development time, stability

and degree of softening.

2-2-3- Bread formulation and production procedure

The following base formulation has used in this work: wheat flour (100 g), water (

56 ml), salt 2g , sugar 8 g, oil 8 g. Glucose oxidase and xylanase were added to the

formulation according to Central Composite Rotational Design (CCRD) (Table1).

The quantities added ranged from 10 ppm to 30 ppm for xylanase, and from 10 to 50

ppm for glucose oxidase. For bread baking, bread making machine have been

used, which carry out all the process operations, mixing, kneading , re-kneading,

fermentation, final proof, baking, in the same room in which aerations parameters

(temperature, time) are strictly controlled relying on the program ,offering the

possibility to correctly compare the obtained results. Steps for making bread with

bread making machine were as follows: The ingredients were mixed for 10 minutes

to form dough and allowed to ferment at 30 ˚C for 180 minutes. First and second

punches were made after 120 and 150 minutes, respectively. Final proofing was

done for 45 minutes at 35˚C.The bread was baked at 232 ˚C for 15 minutes.

2-2-4- Bread evaluation

2-2-4-1- Specific volume

6
Specific volume was determined by seed displacement method (AACC method 10-

05.01)and calculated as the ratio (v/m). Specific volume determination was carried

out 1h after leaving the oven in triplicate.

2-2-4-2- Texture: firmness and springiness

Crumb firmness was determined using AACC method 74-09.01(2010), and an

adaption of this method used to determine springiness according to sangnark and

Noomhorm (2004). The analyses were carried out using a TA-XT2 texturometer

with a 25 kg load (stable Micro systems, Surrey, England) with the p/25 cylindrical

aluminum sensor probe. The parameters established were: test option and mode=

measurement of the compression force, hold until time, pretest speed = 10 mm/s ,

test speed = 1.7 mm/s , posttest speed = 10 mm/s, distance = 40 %, time = 60 s and

auto trigger = 10 g. Fourteen replicates were carried out for each trial.

2-2-4-3- Shape

The shape of the breads was determined according to Bodroza-Solarov.Filipcev,

Kevresan,Mandic, and Simurina (2008). The height and width of the central slice of

the breads were measured using a pachymeter and the shape determined from the

height/ width ratio. A ratio of 0.5 indicates a regular roll shape, a ratio above 0.5

indicates a spherical shape, whilst a ratio below 0.5 indicates a flat shape.

2-2-4-4- Sensory evaluation

Sensory evaluation tests were done by 6 trained judges using the response surface

method, internal and external characteristics were measured.

The sensory scores for external characteristics (volume, crust color, shred and

symmetry of form and crust characteristics) and for internal characteristics (grain,

crumb color, aroma and taste, chew ability and crumb texture) were recorded for

7
each loaf assigned by a panel of trained judges according to the bread score method

developed by the American Institute of Baking and reported by Matz (1960).

These scores was converted into a global concept determined as: very good

(>90), good (80-90), regular(70-80) and detestable(<70) ( Camargo & declor,2009).

2-2-5- Experimental design and statistical analyses

The effect of enzymes additions on bread quality and dough properties were

determined by Response Surface Methodology (RSM) using a central composite

design (CCD). The independent variables were glucose oxidase (10-50 ppm) and

xylanase (10-30ppm). The enzyme levels were selected according to manufacture

recommendations. The responses or dependent variables were: development time,

water absorption, stability and softening degree of dough, specific volume, texture,

shape and thirteen sensory attributes of bread.

The effects of independent variables on the dough properties and bread quality were

studied using CCD, thirteen treatment combinations were generated by statistical

software, Design Expert version 8 (Table 1). Models were developed to relate

independent variables on the dough and bread quality. Table 2 show the coefficients

of the variables in the models and their contribution to the model‘s variation. R2

values were used to judge the adequacy of the models (Table 2). Five central points

made it possible to estimate the pure error of the analyses. The statistical

significance of the terms in the regression equations was examined by ANOVA for

each response and the significance test level was set at 5% (p <0.05).

3. Results and discussions

3-1-Optimization of α-amylase activity

The falling number (FN) of the flour was 603s. According to reference values in the

falling number user manual this result was an indication that the level of natural

8
amylase activity in the flour was naturally low so it was necessary to supplement the

flour with fungal amylase.

Three different doses of amylase enzyme (5, 27 and50 ppm) were added to flour and

fungal α-amylase activity (FFN) was measured. Results determined by FFN were

115(s), 73 (s) and 56(s), respectively. These results were compared with reference

values in the instruction manual.

According to reference values in the user manual for falling number, 5 ppm of

fungal amylase was selected as the optimum dose and added to the flours samples.

Then, rheological properties of the optimized flour were determined on a

farinograph (Table3).

Table (3) shows, results of the farinograph, these results determine that the control

flour (Table 3,Trial 14) had a good value for dough resistance in comparison to

flour samples optimized by α-amylase ( Table3, Trial 15).

Therefore the addition of alpha amylase led to a significant increase in

softening degree of dough. Because polysaccharide degrading enzymes promoted

a similar significant decrease of dough stability. These results are similar to those

obtained by previous investigations (Hyunkim ,Maeda,Morita, 2006), Martinez

Anaya and jimenez (1997, 1998). stated that hydrolytic enzymes acting on

carbohydrates induce a quick response in dough rheology. Alpha amylase

widely modified dough rheology characteristics ,and dough extensibility

reduction promoted (Whitehurst et al., 2010).

To overcome such a problem, in this study the glucose oxidase and xylanase

enzymes has been added to the flours after addition of optimal dose of fungal α-

amylase. So, in the experimental design related to the optimization of glucose

oxidase and xylanase, there is no combination in the absence of the alpha

9
amylase.Rheology properties of the dough are shown in Table 3. Changes in

farinograph parameters after addition of the combination of glucose oxidase and

xylanase to flour are as follows

3-2- Farinograph parameters after addition of glucose oxidase and xylanase

enzymes to optimized flour by fungal amylase

3-2-1- Dough development time

Development time of dough varied between 1.2 (min) and 3.4 (min) after addition of

the combination of glucose oxidase and xylanase (Table3). Statistical analysis of the

results showed that the enzymes ( glucose oxidase and xylanase) have a significant

influence ( p< 0.05) on this parameter, so a coded model (Table 2) and response

surfaces (Figure 1.a) could be obtained.

According to the response surface figure (Figure 1.a), there is a direct correlation

between the level of glucose oxidase and dough development time but an inverse

relationship between the amount of xylanase and dough development time. The

longer development time of dough were observed with the higher

concentrations of glucose oxidase and the lower concentration of xylanase .

The reason of this is because glucose oxidase creates disulfide bonds in the

bread dough, and disulfide bonds increase dough stability and development time

(Rasiah, Sutton,Low,Lin,Gerrard,2005).

Xylanase decrease the development time due to a reduction of the viscosity

in dough , caused by the depolymerization of arabinoxylans (Rouau, Daviet ,

Tahir ,Cherel, & Saulnier, 2006).

3-2-2- Degree of Softening

Evaluation for degree of dough softening after 12 min and degree of dough softening

after 10 min, varied between (60-159 FU) and (93–136 FU), respectively (Table 3).

10
The combination of glucose oxidase and xylanase enzymes influenced dough

softening, and variation in values for this response was explained from the

variation in concentration of these enzymes (p< 0.05).

Results demonstrate that xylanase and glucose oxidase concentrations at the

central values (20 ppm for xylanase and 30 ppm for glucose oxidase) (Figure 1.b,

Table3) the softening degree was higher and lower concentrations of these

enzymes caused a decrease in softening of the dough. That is, in both cases of a

lack and an excess of these enzymes affected the increase in softening degree

of the dough (Figure 1.b).So an optimum dose for both glucose oxidase and

xylanase was determined , as shown by the response surface figure.

According to the response surface figure (Figure 1.b), doses greater than 20ppm of

xylanase and 30 ppm of glucose oxidase induced an increased effect on dough

softening and doses less than 20ppm of xylanase and 30 ppm of glucose oxidase

caused a decrease in the degree of softening.

According to Maat , Roza, Verbakel, Stem, Santos Da Silva, Bosse (1992), the

impact of xylanase in terms of reducing softening degree was due to

redistribution of pentosan and water in the gluten network,an increase in the

volum of gluten made it more extensible , and resulted in a lower evaluation for

softening degree.

But at concentrations greater than 20 ppm, xylanase break down soluble pentosans.

Martinez Anaya and Jimenez (1998) reported that starch and non-starch

hydrolyzing enzymes result in release of free water and change the soluble

fraction of dough. These effects were apparent immediately after mixing and

continued during resting of dough , which changed viscoelastic properties of

dough. Also, according to Figure1.b, at concentrations less than 30 ppm of glucose

11
oxidase, the softening degree decreased. The reason for this is due to the hydrogen

peroxide produced during glucose oxidase reaction. It promotes the formation of

disulfide linkages in gluten protein (Gujral & Rosell,2004, Primo Martin et

al.,2005, vemulapalli & Hoseney, 1998). Covalence bonds in dough make it strong

and softening degree of dough will reduce .But adding too much glucose oxidase

(doses greater than 30 ppm) induced a gluten network with a more discontinuous

protein matrix structure that is completely different from its original orientation. The

number and size of pores in the dough was greatly increased and some of the

pores become stacked giving a disrupted structure to the dough. This disrupted

dough structure affected caused irregularity in its ability to retain water. The gluten

matrix that was produced after treatment with the highest glucose oxidase dosage

was less uniform and more likely to have poor ability to hold gas (Bonet , Rosell,

Caballero, Gomez, Munuera, Liuch, 2006).

3-2-3-Water absorption

Water absorption can be explained mathematically from the variation of glucose

oxidase presented by a codified model (Table2).The responses surfaces model

(figure 1.c) shows that , an increase in glucose oxidase concentration contributed

to an increase in water absorption at any concentration of xylanase.

Glucose oxidase affected the gluten network by making it stronger, so it was able to

absorb much more water (Rasiah, Sutton, Low, Lin, Gerrard, 2005).

Xylanase addition had no effect on water absorption and this study indicated that

water absorption is independent of xylanase concentration.

3-2-4-Stability

Evaluation for dough stability varied from 2.15 min to 5.6 min after additions of

glucose oxidase and xylanase to optimized flour (Table3). Enzymes studied in these

12
tests had a significant influence ( p<0.05) on this parameter, so a coded model

(Table2) and response surfaces ( Figure 1.d ) could be obtained.

According to figure (1.d), addition of glucose oxidase to flour resulted in an

increased evaluations for dough stability because glucose, preferably in the β form,

is oxidized by glucose oxidase to form gluconolacton , in this reaction hydrogen

peroxide is formed. One explanation of the reaction mechanism is that hydrogen

peroxide, in the presence of endogenous peroxidase, naturally occurring in flour,

promotes oxidation of sulfhydryl groups to disulfide bridges in a gluten

network. The increased strength of a gluten network produces increased dough

stability (Rasiah & etal, 2005) .

According to Figure 1.d, the effect of xylanase on dough stability is dose dependent.

At concentrations greater than 20 ppm of xylanase, dough can collapse due to

reduced viscosity in dough aqueous extract caused by the pronounced

depolymerization of arabinoxylans(Rouau et al., 2006).

At concentration of less than 20 ppm, xylanase increases dough stability due either

the removal of or prevention of the negative effect of non-water extractable

arabinoxylans on gluten yield (Wang,Oudgenoeg,Vliet &Hamer,2003). Also, the

effect of the interaction among the glucose oxidase and xylanase was verified.

Glucose oxidase could affect the gluten disulfide bridges and the oxidation of

arabinoxylans via fluoric acid. In the absence of xylanase, high molecular weighted

arabinoxylans are intercrossed. In the presence of xylanase, there is either the

intercrossing of small fragments of arabinoxylans and high molecular weight

fragments, which led to larger polymers. Thus, an elastic gel could be formed, which

would greatly increase the water binding capacity of arabinoxylans, and could

increase dough stability (Autio, 2006; Forman, 2004; Primo Martin et al., 2005).

13
3–3- Specific volume

Specific volume of toast bread was determined on the day of processing and after

cooling. Values for specific volume of bread produced in these tests after enzyme

additions varied from 3 to 3.8 ml/g, with2.1 ml/g for the control and 2.7 ml/g for

flour optimized by fungal amylase.

The results obtained indicated that α- amylase improved the specific volume. α-

Amylase degraded damaged starch into small dextrins, thus allowing the yeast to

work continuously during dough fermentation and this resulted in improved

bread volume. Also amylase functionality under increased specific volume maybe

related to reduced dough viscosity during starch gelatinization, thus prolonging the

rising process in the oven (Goesaert, Slade, Levin & Delcour, 2009).

After adding glucose oxidase and xylanase to the flour with optimum fungal amylase

activity, the values obtained for the thirteen experimental tests were very close to

each other. For these variables, models could not be established as a function of two

enzymes tested in this study, since no significant linear or quadratic effect was

present (p<0.05).

3-4- Texture: firmness and springiness

The crumb firmness of bread can be explained mathematically by a codified model

from the variation of the enzymes (Table2). It can be seen from the response surfaces

(Fig 2.a) that with a fixed concentration of amylase, an increase in glucose oxidase

concentration contributed to an increase in firmness and as the xylanase

concentration increased, firmness gradually decreased. The value obtained for

crumb springiness for the trials of the experimental design were very close to one

another ( Table3).Due to the minimal differences between the values mathematical

14
models could not be obtained for these responses as a function of the variation in the

enzymes studied. The mean value was 50.27.

3-5- Shape

Table 3 shows the evaluations obtained for bread shape. The values obtained by

these tests were very closely related to one another. For these variables models could

not be established as a function of the enzymes studied. As there was no linear or

quadratic effect of interactions between the variables determined with significance

(p< 0.5). This indicates that none of tested enzymes interfered affected these

evaluations determining that quality parameter of shape is independent of amounts

of glucose oxidase and xylanase additions.

All bread in the tests had better evaluations than did the control (Table3) , Caballero

, Gomez and Rosell (2007) showed that glucose oxidase and xylanase produced

bread with a better shape, and Hilhorst , Dunnewind, Orsel , Stegeman , Vliet ,

Gruppen (1999) verified that the combined use of glucose oxidase and xylanase in

conventional crusty bread provided a better shape (base not flat) than individually

employed enzymes.

3-4-Sensory evaluation

Evaluation of the sensory properties of the breads after adding α- amylase enzyme

showed that the use of amylase can improve some bread properties such as

volume, crust color, aroma and taste , chewiness, texture and symmetry of form

while the other parameters did not show significant differences . Sensory evaluation

results after adding combination of glucose oxidase and xylanase showed that these

enzymes can improve volume, symmetry of form, crust characteristics, and grain

and texture while the other parameters did not show significant differences .

Statistical significant parameters, after adding the combination of glucose oxidase

15
and xylanase such as crust characteristics, grain and texture of breads will be

described below.

3-4-1- Crust characteristics

Results of statistical analyses (Table 2 and Table 3) indicated that xylanase addition

had no significant effect on bread crust but about glucose oxidase, there is an inverse

relationship between this enzyme and bread crust (Fig 2.b). The negative impact of

glucose oxidase on crust quality is due to the oxidation effect of glucose oxidase

enzyme that gave dough and resulting bread a flaky quality.

3-4-2- Bread grain

Bread grain analyses (Table2 and Table3) indicated that only the xylanase enzyme

was effective on this parameter, results demonstrated an inverse relationship

between xylanase enzyme and bread grain (Fig 2.c). This can be explained on the

basis of higher release of fermentable sugars in xylanase supplemented dough and

thereby a higher rate of carbon dioxide evolution by the yeast. As a consequence of

the hydrolytic action of xylenes, free sugars such as pentose could be released,

which might be used by microorganisms for fermentation. Excessive production of

carbon dioxide leads to the formation of large pores in bread and has an effect on

bread grain.

3-4-3-Texture

The enzymes studied in these work had a significant influence ( p<0.05) on the

parameter of texture, so a coded model (Table2) and response surfaces ( Figure 2.d

) has been obtained. Results showed that both glucose oxidase and xylanase

improved bread texture. It has been described that xylanase enzyme exerts an

important role in the formation of gluten and dough networks xylanase hydrolyses

water insoluble arabinoxylans causing a decrease in molecular weight of these

16
polymers and makes them water soluble. Water soluble arabinoxylans have a

positive effect on dough properties and bread quality (Martinez-Anaya and Jimenez,

1997).glucose oxidase increases water absorption of dough and in this way it

prevents the texture of from becoming pasty and improves its quality.

Gomez,Del Real, Rosell,Ronda,Blance,Caballero, (2004) reported that products

elaborated with enzymes exhibit a marked improvement in crumb structure.

3- 4-4- Total score

All evaluations for bread from assays in these tests were determined as better those

of the control. The best total scores 84, (good according to Camargo, 1987) were

obtained for bread containing 50 ppm glucose oxidase and 20 ppm xylanase. The

specific characteristics use to determine scores for sensory evaluation were those of:

volume, color of crust, crumb structure and crumb texture. This study showed

that glucose oxidase and xylanase enzymes have a beneficial effect on the sensory

attributes of bread score.

4. Conclusions

It can be concluded that adding a combination of glucose oxidase and xylanase

enzymes to weak flour for bread dough can be applied as a corrective action to

reduce problems in dough caused by the use of amylase enzymes. This study showed

that the effects of glucose oxidase and xylanase are dose dependent and it is

necessary to determine optimal doses of enzymes before adding them to the flour. In

this work optimal dose of amylase, glucose oxidase and xylanase enzymes were 5

ppm, 30 ppm and 20 ppm, respectively. Combination of optimum levels of the three

enzymes results in dough with low stickiness and a bread with higher specific

volume, higher quality texture, better shape and higher total score in sensory

evaluation test than control (without enzyme).

17
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Figure caption:

Fig 1. Response surface plot of dough properties as a function of glucose oxidase

and xylanase enzymes.

Fig 2. Response surface plot of bread quality as a function of glucose oxidase and

xylanase enzymes.
 Tables

Table1. Central composite design for the glucose oxidase and xylanase addition.
Trials A(ppm)a GO(PPM)b X(PPM)c

1 5 6 20
2 5 10 10
3 5 10 30
4 5 30 20
5 5 30 20
6 5 30 20
7 5 30 8
8 5 30 20
9 5 30 20
10 5 30 32
11 5 50 30
12 5 50 10
13 5 53 20
14d 0 0 0
15e 5 0 0

a
A= alpha amylase , bGO= glucose oxidase , cX= xylanase , dtrial 14 is control sample . e trial 15 is corrected flour
by alpha amylase
 Table 2. Coded models for quality parameters as a function of the quantities of the glucose oxidase and
xylanase enzymes (the coded values of the independent variables must be used)

Parameters Coded model

Dough Development time 1.74 + .46 GOa + .4 GO 2 - .33 Xb+ 4.56 X2- .5 GO X
. R2c= .85
parameters
Water absorption 59.28 + .57 GO + .066 X
R2= .62

Softening degree 64.52 – 10.51 GO + 22.89 GO2+ 1.95 X + 7.44X2+

(after 10 min) 2.63 GO X R2 = .98
Softening degree 99.55–4.57GO +18.89 GO2+ .015X + 4.49X2-2GO X
(12 min after max) R2 = .92
Bread Firmness 197+73.83GO-27.57X-14.49GOX+9.97GO2-8.53X2
R2=.98
parameters

Sensory Crust characteristic 2.08-.24GO+.032X

evaluation Grain 7.18+.25 GO+.3GO2-.62X+ .24 X2-.4 GOX

Texture 10.02+.12 GO+.7GO2-.1.68X+ 1.17X2-.4 GOX

Total score 72.26+.25 GO+.58GO2- 2.82X- 3.56X2- 2.16 GOX

a
GO= coded value (- α to + α)of the quantities of glucose oxidase;b X= coded value (- α to + α)of the quantities of
xylanase. cR2= regression coefficient
 Table 3 .Quality parameters in relation to the enzymes glucose oxidase and xylanase and alpha amylase

Trils assay
1 2 3 4 5 6 7 8 9 10 11 12 13 14a 15b

Dough Dough 1.5 1.7 1.9 1.7 1.8 1.6 1.7 2.45 3.4 1.5 3.1 1.7 1.7 1.45 1.2
parameters development
time(min)
Water 57.85 58.8 59.4 59.4 59.2 59.1 59.4 59.3 59.6 59 60 59.7 59.85 57.8 54.65
absorption(lit)
Stability 3.6 3.1 3.35 5.6 5.1 4.8 4.9 5.3 5.1 4.05 4.6 5.25 4.85 3.1 2.15
(min)
Degree 106.5 108 106.5 60 68 65 71.5 63 68 75 88.5 79.5 85 106 159.5
softening
after 10
min(fu)
Degree 136.5 121.5 127 93 103 99 111 98 100 108 115 117.5 123.5 131 172
softening
after 20
min(fu)
Bread Specific 3.6 3 3.5 3.07 3.08 3.1 3.23 3.06 3.11 3.3 3.3 3.2 3.04 1.9 2.75
parameters volume
(ml/g)
Firmness(N) 114 122 101 198 200 197 213 202 199 129 248 351 302 119 96

Springiness 50.87 49.9 50.2 50.53 50.5 49.8 50.53 50.5 50.42 50.29 50.33 49.1 50.6 47.51 48.9

Shape 7.18 7.21 7.24 7.3 7.3 7.3 7.35 7.3 7.3 7.27 7.22 7.23 7.28 6.11 6.46

Volume 7 7.92 7.05 7.9 7.9 7.9 8.8 7.9 79 7.08 6.92 9 6.9 4.1 5.5
Sensory
evaluation Symmetry of 1.58 1.17 1.17 1.5 1.5 1.5 1.55 1.5 1.5 1.92 2.3 2.63 1.08 .83 1
form
Crust 2.3 2.5 2.3 1.7 1.7 1.7 1.75 1.7 1.7 2 1.7 1.9 .097 1.6 1.4
characteristic
Grain 7.17 8.02 7.13 7.14 7.14 7.14 8 7.14 7.14 7.33 6.35 9 8.25 7.1 7.5

Texture 7.9 13 8.9 10 10 10 12.4 10 10 11 7.3 13 10 4.8 5.9

Total score 73.5 78.3 72.9 72.1 72.1 72.1 77.6 72.1 72.1 77.4 70 84 72.6 49.3 61

a
trial 14 is control sample . b trial 15 is corrected flour by alpha amylase
 Figures

Figure 1

Fig1.a Fig1.b

Fig1.c

Fig 1.e
Fig1.d
Figure 2:

Fig 2.a Fig 2.b

Texture

Fig 2.c Fig 2.d

Highlights

 The main enzymes used in bakery products are amylases.

 Adding amylase enzymes lead to decrease in rheological properties of dough .

 Using glucose oxidase and xylanase remove dough problems caused by amylases .

 It is necessary to determine optimal dose of enzymes before adding them to the flour.